Microorganism modified for the production of 1,3-propanediol

Abstract

The invention relates to a modified microorganism for the production of PDO from a carbon substrate wherein the microorganism includes a three-step metabolic pathway including a first step of conversion of 2,4-dihydroxybutyrate (DHB) to obtain 2-oxo-4-hydroxybutyrate (OHB) by an enzyme having 2,4-DHB dehydrogenase activity, a second step of decarboxylation of the OHB to obtain 3-hydroxypropionaldehyde by an enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity, and a third step of reduction of the obtained 3-hydroxypropionaldehyde to obtain PDO with an enzyme having 3-hydroxypropionaldehyde reductase activity and the genes enabling the microorganism for the synthesis of DHB.

The invention claimed is: 
     
       1. A modified microorganism for producing 1,3-propanediol (PDO) from a carbon substrate, the microorganism comprising:
 a pathway for synthesis of 2,4-dihydroxybutyrate (DHB); and 
 a three-step metabolic pathway comprising:
 conversion of DHB to obtain 2-oxo-4-hydroxybutyrate (OHB) by an enzyme having 2,4-DHB dehydrogenase activity, 
 decarboxylation of the OHB to obtain 3-hydroxypropionaldehyde by an enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity, and 
 reduction of the obtained 3-hydroxypropionaldehyde to obtain PDO with an enzyme having 3-hydroxypropionaldehyde reductase activity, 
 
 wherein the modified microorganism has been transformed by the introduction of at least three genes respectively encoding the enzyme having DHB dehydrogenase activity, the enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity, and the enzyme having 3-hydroxypropionaldehyde reductase activity. 
 
     
     
       2. The modified microorganism of  claim 1 , wherein the pathway for the synthesis of DHB is from malate. 
     
     
       3. The modified microorganism according to  claim 2 , wherein the microorganism has been further modified by the introduction of genes encoding:
 a malate kinase catalyzing the transformation of malate into 4-phospho-malate, 
 a malate semialdehyde dehydrogenase catalyzing the transformation of 4-phospho-malate into malate-4-semialdehyde, and 
 a malate semialdehyde reductase catalyzing the transformation of malate-4-semialdehyde into 2,4-DHB. 
 
     
     
       4. The modified microorganism of  claim 1 , wherein the enzymes are encoded by an endogenous or a heterologous gene. 
     
     
       5. The modified microorganism of  claim 1 , wherein the enzyme having 2,4-DHB dehydrogenase activity is an enzyme having lactate dehydrogenase or malate dehydrogenase activity. 
     
     
       6. The modified microorganism of  claim 1 , wherein the enzyme having 2,4-DHB dehydrogenase activity is obtained by at least one mutation of an enzyme, said mutation improving activity and/or substrate affinity of the enzyme for DHB. 
     
     
       7. The modified microorganism of  claim 5 , wherein the enzyme having 2,4-DHB dehydrogenase activity is a gene product encoded by a gene selected from the group consisting of IdhA from  Lactococcus lactis , lldD from  Escherichia coli , lldD from  E. coli  carrying a mutation at position Val108 (by reference to SEQ ID No. 122), mdh from  E. coli , mdh from  Bacillus subtilis , and mdh from  E. coli  carrying a mutation in at least one position selected from the group consisting of (by reference to SEQ ID No. 124): Ile12, Arg81, Lys82, Met85, Asp86, Val93, Ile117, Gly179, Thr211, and Met227 (by reference to SEQ ID No.126). 
     
     
       8. The modified microorganism of  claim 7 , wherein the enzyme having 2,4-DHB dehydrogenase activity is:
 encoded by a polynucleotide having the sequence of SEQ ID No. 119, SEQ ID No. 121, SEQ ID No. 153, SEQ ID No. 155, SEQ ID No. 157, SEQ ID No. 159, SEQ ID No. 161, SEQ ID No. 163, SEQ ID No. 165, SEQ ID No. 167, SEQ ID No. 169, SEQ ID No. 171, SEQ ID No. 173, or any sequence sharing a homology of at least 50% with the sequence of SEQ ID No. 119, SEQ ID No. 121, SEQ ID No. 153, SEQ ID No. 155, SEQ ID No. 157, SEQ ID No. 159, SEQ ID No. 161, SEQ ID No. 163, SEQ ID No. 165, SEQ ID No. 167, SEQ ID No. 169, SEQ ID No. 171, or SEQ ID No. 173, and/or 
 a polypeptide having the sequence of SEQ ID No. 120, SEQ ID No. 122, SEQ ID No. 154, SEQ ID No. 156, SEQ ID No. 158, SEQ ID No. 160, SEQ ID No. 162, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 168, SEQ ID No. 170, SEQ ID No. 172, SEQ ID No. 174, or any sequence sharing a homology of at least 50% with the sequence of SEQ ID No. 120, SEQ ID No. 122, SEQ ID No. 154, SEQ ID No. 156, SEQ ID No. 158, SEQ ID No. 160, SEQ ID No. 162, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 168, SEQ ID No. 170, SEQ ID No. 172, or SEQ ID No. 174. 
 
     
     
       9. The modified microorganism of  claim 1 , wherein the enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity is an enzyme having a 2-keto acid decarboxylase activity. 
     
     
       10. The modified microorganism of  claim 1 , wherein the enzyme having 4-hydroxybutyrate decarboxylase activity is obtained by at least one mutation of an enzyme, said mutation improving activity and/or substrate affinity of the enzyme for OHB. 
     
     
       11. The modified microorganism of  claim 9 , wherein the enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity is a gene product encoded by a gene selected from the group consisting of PDC1, PDC5, PDC6, ARO10, and THI3 genes from  Saccharomyces cerevisiae ; kivD and kdcA genes from  Lactococcus lactis ; pdc gene from  Clostridium acetobutylicum ; PDC2 and PDC3 genes from  Arabidopsis thaliana ; PDC1, PDC2, and ARO10 genes from  Pichia stipitis ; pdc gene from  Zymomonas mobilis ; sucA gene from  Escherichia coli ; dxs gene from  Escherichia coli ; pdc gene from  Z. mobilis  carrying a mutation in at least one position selected from the group consisting of: Tyr290, Trp392, Gly413, and Ile476 (by reference to SEQ ID No.128); and kdcA gene from  L. lactis  carrying a mutation in at least one position selected from the group consisting of: Gln377, Phe381, Phe382, Gly402 Val461, Ile465, and Phe542 (by reference to SEQ ID No.130). 
     
     
       12. The modified microorganism of  claim 11 , wherein the enzyme having 4-hydroxybutyrate decarboxylase activity is
 encoded by a polynucleotide having the sequence of SEQ ID No. 129, SEQ ID No. 127, SEQ ID No. 207, SEQ ID No. 189, SEQ ID No. 191, SEQ ID No. 193, SEQ ID No. 195, SEQ ID No. 197, or any sequence sharing a homology of at least 50% with the sequence of SEQ ID No. 129, SEQ ID No. 127, SEQ ID No. 207, SEQ ID No. 189, SEQ ID No. 191, SEQ ID No. 193, SEQ ID No. 195, or SEQ ID No. 197, and/or 
 a polypeptide having the sequence of SEQ ID No. 130, SEQ ID No. 128, SEQ ID No. 208, SEQ ID No. 190, SEQ ID No. 192, SEQ ID No. 194, SEQ ID No. 196, SEQ ID No. 198, or any sequence sharing a homology of at least 50% with the sequence of SEQ ID No. 130, SEQ ID No. 128, SEQ ID No. 208, SEQ ID No. 190, SEQ ID No. 192, SEQ ID No. 194, SEQ ID No. 196, or SEQ ID No. 198. 
 
     
     
       13. The modified microorganism of  claim 1 , wherein the enzyme having 3-hydroxypropionaldehyde reductase activity is an enzyme having hydroxyaldehyde reductase activity, alcohol dehydrogenase activity, lactaldehyde reductase activity, or methylglyoxal reductase activity. 
     
     
       14. The modified microorganism of  claim 13 , wherein the enzyme having 3-hydroxypropionaldehyde reductase activity is:
 a gene product encoded by a gene selected from the group consisting of yqhD, fucO, dkgA, and dkgB genes from  Escherichia coli , dhaT gene from  K. pneumoniae , and ADH1 and ADH2 genes from  Saccharomyces cerevisiae , or 
 an enzyme having 3-hydroxypropionaldehyde reductase activity obtained by at least one mutation of an enzyme, said mutation improving activity and/or substrate affinity of the enzyme for 3-hydroxypropionaldehyde. 
 
     
     
       15. The modified microorganism according to  claim 1 , wherein the production of PDO is enhanced. 
     
     
       16. The modified microorganism according to  claim 1 , wherein the 2,4-dihydroxybutyrate dehydrogenase, 2-oxo-4-hydroxybutyrate decarboxylase, and/or 3-hydroxypropionaldehyde reductase activities, and/or enzymatic activities allowing the synthesis of DHB are enhanced. 
     
     
       17. The modified microorganism of  claim 1 , being a bacterium, a yeast, or a fungus. 
     
     
       18. The modified microorganism of  claim 1 , wherein the expression of:
 at least one enzymatic activity selected from the group consisting of phosphoenolpyruvate carboxylase, phosphoenol pyruvate carboxykinase, isocitrate lyase, pyruvate carboxylase, and hexose symporter permease is increased, and/or 
 at least one enzymatic activity selected from the group consisting of lactate dehydrogenase, alcohol dehydrogenase, acetate kinase, phosphate acetyltransferase, pyruvate oxidase, isocitrate lyase, fumarase, 2-oxoglutarate dehydrogenase, pyruvate kinase, malic enzyme, phosphoglucose isomerase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, pyruvate-formate lyase, succinic semialdehyde dehydrogenase, sugar-transporting phosphotransferase, ketohydroxyglutarate aldolase, homoserine-O-succinyl transferase, homoserine kinase, diaminopimelate decarboxylase, and methylglyoxal synthase is decreased. 
 
     
     
       19. The modified microorganism according to  claim 17 , wherein the modified microorganism is  Escherichia coli  that:
 overexpresses at least one gene selected from the group consisting of ppc, pck, aceA, galP, asd, thrA, metL, and lysC from  E. coli , and pycA from  L. lactis , and/or 
 has at least one deleted gene selected from the group consisting of IdhA, adhE, ackA, pta, poxB, focA, pfIB, sad, gabABC, sfcA, maeB, ppc, pykA, pykF, mgsA, sucAB, ptsl, ptsG, pgi, fumABC, aldA, lldD, icIR, metA, thrB, lysA, and eda. 
 
     
     
       20. A method of production of PDO comprising:
 contacting the modified microorganism of  claim 1  with a carbon substrate in an appropriate culture medium, and 
 recovering PDO from the culture medium. 
 
     
     
       21. The method of  claim 20 , wherein the PDO is further purified. 
     
     
       22. A modified microorganism for producing 1,3-propanediol (PDO) from a carbon substrate, the microorganism comprising:
 a pathway for synthesis of 2,4-dihydroxybutyrate (DHB); and 
 a three-step metabolic pathway comprising:
 conversion of DHB to obtain 2-oxo-4-hydroxybutyrate (OHB) by an enzyme having DHB dehydrogenase activity, 
 decarboxylation of the OHB to obtain 3-hydroxypropionaldehyde by an enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity, and 
 reduction of the obtained 3-hydroxypropionaldehyde to obtain PDO with an enzyme having 3-hydroxypropionaldehyde reductase activity, 
 
 wherein: 
 the gene encoding the enzyme having DHB dehydrogenase activity is selected from the group consisting of IdhA from  Lactococcus lactis , lldD from  Escherichia coli , lldD from  E. coli  carrying a mutation at position Val108 (by reference to SEQ ID No. 122), mdh from  E. coli , mdh from  Bacillus subtilis , and mdh from  E. coli  carrying a mutation in at least one position selected from the group consisting of (by reference to SEQ ID No. 124): Ile12, Arg81, Lys82, Met85, Asp86, Val93, Ile117, Gly179, Thr211, and Met227 (by reference to SEQ ID No.126); 
 the gene encoding the enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity is selected from the group consisting of PDC1, PDC5, PDC6, ARO10, and THIS genes from  Saccharomyces cerevisiae ; kivD and kdcA genes from  Lactococcus lactis ; pdc gene from  Clostridium acetobutylicum ; PDC2 and PDC3 genes from  Arabidopsis thaliana ; PDC1, PDC2, and ARO10 genes from  Pichia stipitis ; pdc gene from  Zymomonas mobilis ; sucA gene from  Escherichia coli ; dxs gene from  Escherichia coli ; pdc gene from  Z. mobilis  carrying a mutation in at least one position selected from the group consisting of: Tyr290, Trp392, Gly413, and Ile476 (by reference to SEQ ID No.128); and kdcA gene from  L. lactis  carrying a mutation in at least one position selected from the group consisting of: Gln377, Phe381, Phe382, Gly402 Val461, Ile465, and Phe542 (by reference to SEQ ID No.130); and 
 the enzyme having 3-hydroxypropionaldehyde reductase activity is:
 a gene product encoded by a gene selected from the group consisting of yqhD, fucO, dkgA, and dkgB genes from  Escherichia coli , dhaT gene from  K. pneumoniae , and ADH1 and ADH2 genes from  Saccharomyces cerevisiae , or 
 an enzyme having 3-hydroxypropionaldehyde reductase activity obtained by at least one mutation of an enzyme, said mutation improving activity and/or substrate affinity of the enzyme for 3-hydroxypropionaldehyde. 
 
 
     
     
       23. The method according to  claim 22 , wherein:
 the enzyme having DHB dehydrogenase activity is Ec-Mdh R81A; 
 the enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity is Zm-Pdc; and 
 the enzyme having 3-hydroxypropionaldehyde reductase activity is Ec-YqhD.